This project is focused on the aerobic and non-aerobic processes that create the peptide and protein- derived quino-cofactors, pyrroloquinoline quinone (PQQ) and trihydroxyphenylalanine quinone (TPQ). The described studies explore a cohort of reactions that are characterized by both unprecedented or highly unusual mechanisms and structure function relationships. PQQ biosynthesis is one of the least well understood among the pathways that lead to enzymatic cofactors, and each of the five conserved open reading frames that encode for essential proteins in PQQ production is being studied. These include the peptide substrate (PqqA), a likely oxygenase (PqqB,) a radical SAM enzyme (PqqE), and a protein with, as yet, no defined function (PqqD). In addition to the planned studies on isolated enzymes, the importance of protein/protein complex formation will undergo detailed investigation. These studies are expected to reveal how the PQQ pathway is able to integrate reactions that either must be anaerobic (PqqE) or require the uptake of molecular oxygen (PqqC and PqqB). PqqC is an oxidase that functions in the absence of any metal or organic cofactor, catalyzing the final step in PQQ formation via an eight-electron, eight-proton oxidation. We have developed an acid quench assay that allows us to characterize many of the chemical intermediates that occur along the PqqC reaction path. This new assay is being used in conjunction with site-specific mutagenesis, to examine the role of specific amino acid side chains (i) in generating an O2 binding/reactivity pocket, (ii) in the stepwise abstraction of eight electrons and protons from the substrate, and (iii) in the creation of a pathway that permits the abstracted protons to be "guided" toward the active site oxidant. Structural studies of the PqqC/product complex implicate a large conformational change that controls O2 reactivity, and the use of substrate itself in the intra-molecular proton transfer conduit.
The final aim of this project focuses on the biogenesis of TPQ, using an expressed, precursor yeast copper amine oxidase. In contrast to PQQ production, TPQ is formed via a self-processing mechanism. These studies use an unnatural amino acid, 4-amino-phenylalanine (p-AF) that has been successfully inserted at three key positions within the enzyme active site. The properties of p-AF, in relation to the replaced tyrosines, are uncovering important features of both cofactor biosynthesis and enzymatic catalysis.

Public Health Relevance

We are investigating post-translational processes that produce the quinocofactors, pyrroloquinoline quinone (PQQ, a bacterial growth factor) and trihydroxyphenylalanine quinone (TPQ, linked to inflammatory response(s) in mammalian vasculature and adipose tissue). Non-canonical aspects of enzyme function are highlighted in these studies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Barski, Oleg
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University of California Berkeley
Schools of Arts and Sciences
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Klinman, Judith P; Bonnot, Florence (2014) Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ. Chem Rev 114:4343-65
Klema, Valerie J; Solheid, Corinne J; Klinman, Judith P et al. (2013) Structural analysis of aliphatic versus aromatic substrate specificity in a copper amine oxidase from Hansenula polymorpha. Biochemistry 52:2291-301
Shen, Yao-Qing; Bonnot, Florence; Imsand, Erin M et al. (2012) Distribution and properties of the genes encoding the biosynthesis of the bacterial cofactor, pyrroloquinoline quinone. Biochemistry 51:2265-75
Shen, Sam H; Wertz, Diana L; Klinman, Judith P (2012) Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies. PLoS One 7:e29270
RoseFigura, Jordan M; Puehringer, Sandra; Schwarzenbacher, Robert et al. (2011) Characterization of a protein-generated O? binding pocket in PqqC, a cofactorless oxidase catalyzing the final step in PQQ production. Biochemistry 50:1556-66
Oyeyemi, Olayinka A; Sours, Kevin M; Lee, Thomas et al. (2010) Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency. Proc Natl Acad Sci U S A 107:10074-9
Wecksler, Stephen R; Stoll, Stefan; Iavarone, Anthony T et al. (2010) Interaction of PqqE and PqqD in the pyrroloquinoline quinone (PQQ) biosynthetic pathway links PqqD to the radical SAM superfamily. Chem Commun (Camb) 46:7031-3
Chen, Zhi-Wei; Datta, Saumen; Dubois, Jennifer L et al. (2010) Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity. Biochemistry 49:7393-402
Chang, Cindy M; Klema, Valerie J; Johnson, Bryan J et al. (2010) Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha. Biochemistry 49:2540-50
Puehringer, Sandra; RoseFigura, Jordan; Metlitzky, Moritz et al. (2010) Structural studies of mutant forms of the PQQ-forming enzyme PqqC in the presence of product and substrate. Proteins 78:2554-62

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